PAPS is a sulfate donor which is widely employed in living organisms, including microorganisms, plants, and higher animals. PAPS has been reported to be related to several diseases (e.g., proteoglycan-associated diseases). PAPS plays a very important role in vivo, and could be employed in, for example, the field of medicine.
Known methods for enzymatically synthesizing PAPS include a method in which PAPS is produced from ATP through two-step reaction employing two enzymes (ATPS and APSK) extracted from baker's yeast or rat liver (the below-described formulas 1 and 2).
However, this method produces only a small amount (several milligrams to several tens of milligrams) of PAPS, and requires ATP, which is an expensive substance; i.e., this method is not necessarily considered industrially practical.
(In the aforementioned formulas, ATP represents adenosine 5′-triphosphate; ADP represents adenosine 5′-diphosphate; APS represents adenosine 5′-phosphosulfate; PPi represents inorganic pyrophosphate; and SO42− represents a sulfate ion.)
Therefore, practical production of PAPS requires employment of microorganism-derived enzymes which ensure high productivity and easy handling (first requirement); and establishment of an ATP supply/regeneration system which does not require ATP (i.e., an expensive substance) (second requirement).
In connection with the first requirement, Onda, et al., have reported that PAPS can be efficiently produced through two-step reaction employing two highly thermostable enzymes (ATPS and APSK) derived from a bacterium belonging to the genus Bacillus or Thermus (Patent Documents 1 to 3).
When the method of Onda, et al., is carried out, ATPS and APSK must be purified from a suspension of disrupted thermotolerant bacterium cells. However, the method involves problems in that the amounts of these enzymes contained in the bacterial cells are small, and thus large amounts of the cells must be cultured, and that purification of these enzymes requires a very intricate process.
Meanwhile, in connection with the second requirement, Ibuki, et al., have reported that a method in which a PAPS synthesis system is linked to an ATP regeneration system including acetyl phosphate and acetate kinase, and ADP produced through APSK reaction is converted into ATP for recycling (Non-Patent Document 1).
However, this method still employs ATP (i.e., an expensive substance) as a substrate at an early stage of reaction, and employs a large amount of acetyl phosphate (i.e., an expensive substance) as a phosphate donor for ATP regeneration. Therefore, the method has not yet been put into practice.
Conventionally known inexpensive phosphate donors include polyphosphate (Poly Pn). Known ATP supply/regeneration systems employing polyphosphate and adenosine 5′-monophosphate (AMP) include a system represented by the below-described formulas (3) and (4) and utilizing the cooperative effect of polyphosphate kinase (PPK) and adenylate kinase (ADK) (Patent Document 4); and a system represented by the below-described formulas (5) and (6) and employing polyphosphate:AMP phosphotransferase (PAP) and ADK (Patent Document 5).
(In the aforementioned formulas, ATP represents adenosine 5′-triphosphate; ADP represents adenosine 5′-diphosphate; AMP represents adenosine 5′-monophosphate; Poly Pn represents polyphosphate; and n represents an integer.)
There has already been reported a method in which the ATP supply/regeneration system utilizing the PPK-ADK cooperative effect is applied to enzymatic synthesis of PAPS (Patent Document 6). However, detailed analysis of this method has revealed that the amount of PAPS produced is at most about 5 mM. Extensive studies on the cause of such low yield have shown that the ATP supply/regeneration system utilizing the PPK-ADK cooperative effect does not function effectively, and difficulty is encountered in maintaining higher concentration of ATP than that of ADP or AMP in the reaction system, resulting in non-efficient PAPS production (see Comparative Example 1 described below).
PPK is considered to function as a polyphosphate synthase in vivo, and the equilibrium of the enzymatic reaction represented by formula (4) is greatly shifted to the side of polyphosphate synthesis (Non-Patent Document 2). As has been indicated, since PPK exhibits low ATP synthesis activity, although the ATP supply/regeneration system employing the enzyme functions well in, for example, a reaction in which the concentration of a substrate is low (i.e., about 5 mM or less), the system fails to function satisfactorily in a reaction in which the concentration of a substrate is high (i.e., more than 10 mM).
There has not yet been reported a case where the ATP supply/regeneration system employing PAP and ADK is applied to enzymatic synthesis of PAPS. However, since ADK completely reversibly catalyzes the reaction represented by formula (6), as described below in Comparative Example 2, the concentration of ATP in the reaction system fails to be maintained high, resulting in non-efficient PAPS production.
Therefore, practical production of PAPS requires establishment of a new potent ATP supply/regeneration system which can maintain higher concentration of ATP than that of ADP or AMP.
Examples of means for establishing such a potent ATP supply/regeneration system include employment of Pseudomonas aeruginosa-derived polyphosphate-driven nucleoside 5′-diphosphate kinase (PNDK). Recently, Pseudomonas aeruginosa-derived PNDK has been identified (Non-Patent Documents 3 and 4), and a gene encoding the enzyme has been obtained (Non-Patent Documents 2 and 5).
Similar to the case of PPK, PNDK exhibits polyphosphate-driven ADP phosphorylation activity. However, the amino acid sequence of PNDK has no homology to that of PPK, and PNDK exhibits remarkably high specific activity; i.e., 100 or more times that of Escherichia coli-derived PPK, and about 1,000 times that of Pseudomonas aeruginosa-derived PPK (Non-Patent Document 2). In contrast to the case of PPK, PNDK exhibits almost negligibly low polyphosphate synthesis activity, and the reaction equilibrium is greatly shifted to the side of ATP synthesis. Therefore, PNDK is considered an ideal enzyme employed for an ATP supply/regeneration system.
However, productivity of the enzyme PNDK in Pseudomonas aeruginosa is low. Therefore, when the enzyme is employed on an industrial scale, a gene for producing the enzyme must be cloned through a genetic recombination technique, and the enzyme must be mass-produced by use of a host such as Escherichia coli. However, in experiments previously performed by the present inventors, even when such a genetic recombination technique was employed, productivity of the enzyme is still low, and thus the enzyme was difficult to put into practice.    Patent Document 1: Japanese Patent No. 3098591    Patent Document 2: Japanese Patent No. 3078067    Patent Document 3: Japanese Patent No. 3029915    Patent Document 4: WO 98/48031    Patent Document 5: WO 03/100056    Patent Document 6: JP-A-2002-78498    Non-Patent Document 1: Nucleic Acids Symp. Ser., 27, 171-172 (1992)    Non-Patent Document 2: Proc. Natl. Acad. Sci. USA, 99, 16684-16688 (2002)    Non-Patent Document 3: Proc. Natl. Acad. Sci. USA, 94, 439-442 (1997)    Non-Patent Document 4: Biochem. Biophys. Res. Commun., 281, 821-826 (2001)    Non-Patent Document 5: Proc. Natl. Acad. Sci. USA, 99, 16678-16683 (2002)